Project Summary/Abstract Major diseases including obesity, the metabolic syndrome, and type 2 diabetes, are associated with insulin resistance and consequently impaired glucose homeostasis. A key to the maintenance of glucose homeostasis under fasting and fed conditions is the proper control of the subcellular distribution of the glucose transporter GLUT4 in muscle and fat cells. Under fasting conditions a low number of GLUT4 at the cell surface limits the uptake of glucose into muscle and fat cells and thus allows the circulating glucose to be available as fuel for the brain. After a meal, in response to insulin, the number of GLUT4 at the cell surface is dramatically increased, through a process known as GLUT4 translocation. This facilitates glucose uptake and promotes the disposal of 80-90% of the ingested glucose into muscle and fat. In addition, GLUT4 translocation is also responsible for increased glucose uptake in skeletal muscles in response to exercise. AS160, a Rab GTPase activating protein (Rab GAP), and its recently identified relative Tbc1d1, play important roles in the regulation of the subcellular distribution of GLUT4, as established in cultured fat and muscle cells and specific skeletal muscles in mice. They are responsible for the efficient intracellular retention of GLUT4 under basal conditions, as well as the release of GLUT4 from retention and translocation of GLUT4 to the cell surface in response to insulin and exercise. The roles of AS160 and Tbc1d1 have not yet been evaluated in vivo. Preliminary data with mice in which AS160 had been deleted (AS160 knockout mice), suggest an essential role for AS160 in the regulation of glucose uptake in adipocytes, but not in skeletal muscles. Tbc1d1 knockout mice are being generated and no preliminary results are yet available from these mice. Considering that Tbc1d1 is expressed at higher levels in some skeletal muscle types, it is possible that Tbc1d1 is the major regulator of GLUT4 translocation in skeletal muscles. Our hypothesis is that AS160 and Tbc1d1 play important and possibly unique roles in the regulation of the subcellular distribution of GLUT4 in different cell types. The specific aims of the proposed research are to determine the roles of AS160 and Tbc1d1 by analyzing glucose homeostasis and the regulation of the subcellular distribution of GLUT4 in muscle and fat cells under basal conditions and in response to insulin and exercise in AS160 knockout, Tbc1d1 knockout, and double AS160/Tbc1d1 knockout mice. Our research will thus elucidate fundamental mechanisms contributing to the maintenance of glucose homeostasis. It will further provide crucial information towards the validation of AS160 and Tbc1d1 as drug targets to improve glucose homeostasis and prevent ensuing complications of insulin resistance.